Estimates of Genetic Differentiation Measured by F(ST) Do Not Necessarily Require Large Sample Sizes When Using Many SNP Markers

Population genetic studies provide insights into the evolutionary processes that influence the distribution of sequence variants within and among wild populations. F_ST is among the most widely used measures for genetic differentiation and plays a central role in ecological and evolutionary genetic studies. It is commonly thought that large sample sizes are required in order to precisely infer F_ST and that small sample sizes lead to overestimation of genetic differentiation. Until recently, studies in ecological model organisms incorporated a limited number of genetic markers, but since the emergence of next generation sequencing, the panel size of genetic markers available even in non-reference organisms has rapidly increased. In this study we examine whether a large number of genetic markers can substitute for small sample sizes when estimating F_ST. We tested the behavior of three different estimators that infer F_ST and that are commonly used in population genetic studies. By simulating populations, we assessed the effects of sample size and the number of markers on the various estimates of genetic differentiation. Furthermore, we tested the effect of ascertainment bias on these estimates. We show that the population sample size can be significantly reduced (as small as n = 4–6) when using an appropriate estimator and a large number of bi-allelic genetic markers (k>1,000). Therefore, conservation genetic studies can now obtain almost the same statistical power as studies performed on model organisms using markers developed with next-generation sequencing.     

On the compositing of samples for qualitative microbiological testing

The introduction of legislative microbiological criteria for foods [Official J L338 (2005) 1] has increased the exposure of enforcement and industrial laboratories to the need to test multiple food samples for organisms such as salmonellae. A consequence has been an increase in the number of organizations, both official and commercial, considering whether it is permissible to composite replicate sample units for the purposes of assessing compliance with the criteria. This note summarizes the statistical and practical aspects of compositing sample units for compliance testing. Provided that the method of choice is 'fit for purpose', then there is no statistical difference between testing, say, 30 x 25 g sample units or 3 x 250 g sample units. However, compositing of sample units should be done only when the method has been demonstrated unequivocally to be sufficiently sensitive to detect potentially lower numbers of target organism(s) in the quantity of composited sample under the conditions of test. Different approaches to compositing of samples are considered.     

Seasonal selection in a freshwater heterotrophic bacterial community

The objective of this study was to determine if a seasonal selection could be demonstrated in the heterotrophic component of a freshwater bacterial community. Surface samples were taken at approximately monthly intervals covering an annual seasonal cycle, and counts were made of the numbers of bacteria capable of growing at each of 10 incubation temperatures from 0° to 45°C at 5°C intervals. Evidence for seasonal selection was provided by a 6°C shift in the mean temperature of the counts from the summer sample to the winter sample. The selection was even more evident when the number of organisms capable of growing at 10°C and those capable of growing at 35°C were compared over the seasonal cycle. The counts at these two incubation temperatures varied inversely to each other. Although a negligible number of organisms from a representative summer sample grew at 10°C, 18% of the organisms from a representative winter sample grew at this temperature. The data of this study indicate that, although seasonal selection does occur, the magnitude of that selection is not great enough to permit the growth of bacteria during the coldest month to approach the levels of growth observed during the summer months. However, the selection appears to be adequate to permit significant activity during the spring and fall transition months.     

Cascading tripartite binomial classification plans to monitor European red mite (Acari,Tetranychidae) through a season; development and evaluation of a new methodology for pest monitoring

A monitoring protocol that schedules future sample bouts based on the outcome of density classification and expected population growth has been developed and applied to monitoring European red mite (Panonychus ulmi Koch) through a growing season. The monitoring protocol is based on concatenating through time tripartite sequential classification sampling plans that use binomial counts in lieu of complete enumeration. Binomial counts are scored positive when the number of organisms (mites) on a sample unit (leaves) exceeds a tally number. At each sample occasion the monitoring procedure leads to one of three possible decisions; intervene when the density is high, sample at the next sample occasion (after one week) when the density is intermediate, and sample at the second next sample occasion (after two weeks) when the density is low. Evaluation of the monitoring protocol under field conditions showed that the protocol with constituent tally 0 binomial count sampling plans was quite successful in timing intervention at the moment when population densities were about to exceed an established threshold that dictated intervention. The performance of this monitoring protocol and another protocol in which constituent sampling plans used binomial counts with a tally number of 4 were compared using simulation. Sampling plans that used a tally number of 4 were more precise than plans that used a tally number of 0. However, the overall performance of the monitoring protocol based on tally 0 sampling plans did not greatly differ from the monitoring protocol based on tally 4 sampling plans. Simulated performance of the tally 0 protocol was corroborated by field evaluation. The monitoring protocol based on tripartite classification required 30 to 45 percent fewer sample bouts than a protocol based on conventional sequential classification at weekly intervals. The monitoring protocol based on tripartite classification was also better able to schedule intervention when needed compared to a protocol based on conventional classification at two week intervals. Using the tally 0 protocol and current thresholds forP. ulmi, cumulative mite density was kept below 300 mite-days per leaf, which is well below levels regarded damaging. A tally 0 protocol with raised thresholds, developed on the basis of this finding, gave the best simulated performance of all protocols evaluated.     

Mathematical consequences of the genealogical species concept

A genealogical species is defined as a basal group of organisms whose members are all more closely related to each other than they are to any organisms outside the group ("exclusivity"), and which contains no exclusive group within it. In practice, a pair of species is so defined when phylogenies of alleles from a sample of loci shows them to be reciprocally monophyletic at all or some specified fraction of the loci. We investigate the length of time it takes to attain this status when an ancestral population divides into two descendant populations of equal size with no gene exchange, and when genetic drift and mutation are the only evolutionary forces operating. The number of loci used has a substantial effect on the probability of observing reciprocal monophyly at different times after population separation, with very long times needed to observe complete reciprocal monophyly for a large number of loci. In contrast, the number of alleles sampled per locus has a relatively small effect on the probability of reciprocal monophyly. Because a single mitochondrial or chloroplast locus becomes reciprocally monophyletic much faster than does a single nuclear locus, it is not advisable to use mitochondrial and chloroplast DNA to recognize genealogical species for long periods after population divergence. Using a weaker criterion of assigning genealogical species status when more than 50% of sampled nuclear loci show reciprocal monophyly, genealogical species status depends much less on the number of sampled loci, and is attained at roughly 4-7 N generations after populations are isolated, where N is the historically effective population size of each descendant. If genealogical species status is defined as more than 95% of sampled nuclear loci showing reciprocal monophyly, this status is attained after roughly 9-12 N generations.     

The detection of irradiated foods using the Direct Epifluorescent Filter Technique

A method was evaluated which has the potential to detect a food sample which has been irradiated. The technique will give an indication of the total number of viable micro-organisms present before irradiation. It is based on the comparison of an aerobic plate count (APC) with a count obtained using the Direct Epifluorescent Filter Technique (DEFT). When the APC of an irradiated sample was compared with the DEFT count on the same sample, the APC was considerably lower than that obtained by DEFT. The count of orange fluorescing cells after irradiation, however, correlated well with an APC of the same sample before irradiation. For the samples examined the DEFT count determined the viable microbial population in the sample before irradiation. The difference between the APC and the DEFT count gave the number of organisms rendered non-viable by the process.     

The detection of irradiated foods using the Direct Epifluorescent Filter Technique

A method was evaluated which has the potential to detect a food sample which has been irradiated. The technique will give an indication of the total number of viable micro-organisms present before irradiation. It is based on the comparison of an aerobic plate count (APC) with a count obtained using the Direct Epifluorescent Filter Technique (DEFT). When the APC of an irradiated sample was compared with the DEFT count on the same sample, the APC was considerably lower than that obtained by DEFT. The count of orange fluorescing cells after irradiation, however, correlated well with an APC of the same sample before irradiation. For the samples examined the DEFT count determined the viable microbial population in the sample before irradiation. The difference between the APC and the DEFT count gave the number of organisms rendered non-viable by the process.     

Accuracy of coalescent likelihood estimates: do we need more sites, more sequences, or more loci?

A computer simulation study has been made of the accuracy of estimates of Theta = 4Nemu from a sample from a single isolated population of finite size. The accuracies turn out to be well predicted by a formula developed by Fu and Li, who used optimistic assumptions. Their formulas are restated in terms of accuracy, defined here as the reciprocal of the squared coefficient of variation. This should be proportional to sample size when the entities sampled provide independent information. Using these formulas for accuracy, the sampling strategy for estimation of Theta can be investigated. Two models for cost have been used, a cost-per-base model and a cost-per-read model. The former would lead us to prefer to have a very large number of loci, each one base long. The latter, which is more realistic, causes us to prefer to have one read per locus and an optimum sample size which declines as costs of sampling organisms increase. For realistic values, the optimum sample size is 8 or fewer individuals. This is quite close to the results obtained by Pluzhnikov and Donnelly for a cost-per-base model, evaluating other estimators of Theta. It can be understood by considering that the resources spent collecting larger samples prevent us from considering more loci. An examination of the efficiency of Watterson's estimator of Theta was also made, and it was found to be reasonably efficient when the number of mutants per generation in the sequence in the whole population is less than 2.5.     

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.     

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.     

A simplified method for the biological assessment of the quality of fresh and slightly brackish water

A simplified method for the biological assessment of the saprobic quality of surface water is described. The method has the advantage that it is unnecessery to determine accurately the species of organisms present in the sample, which must be done when existing saprobic systems are used. The method described here cannot be used when there is a great abundance of a single species and a very strong environmental disturbance must be assumed.     

Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population

Abstract Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively. This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance. A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number. A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number. Received: 29 January 1999; Accepted: 29 April 1999     

Bacteremia in Experimental Tyzzer's Disease of Mice

Bacteremia was observed during the late stage of experimental Tyzzer's disease in mice. The number of organisms in the blood in mice treated with cortisone increased markedly in the infection with highly virulent organisms, whereas bacteremia was of low incidence and less severe in infections with low virulence organisms. The number of organisms in the blood stream was shown to increase linearly during the course of fatal infection attaining a maximum level of 10⁷ organisms per ml blood. The number of organisms in the blood was found to be closely related to the number in the liver when bacteremia was observed. In the peripheral blood, organisms were first detectable when the number of organisms in the liver gained a level of 10⁷ per g tissue, and the subsequent increase in the number of bacteria in the blood was approximately 3 times more rapid than in the liver. The organisms in the blood were comparable to those in the liver morphologically as well as in pathogenicity. Histopathological examination frequently revealed liberation of organisms from liver cells into sinusoids. There was no evidence of significant multiplication of the organisms in organs other than the liver.     

Microbial identification by mass cataloging

Background  

The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using hybridization and other molecular approaches. In their usual format, such assays are based on the presence of unique subsequences in the target RNA and require a prior knowledge of what organisms are likely to be in a sample. They are thus limited in generality when analyzing an unknown sample.     

The choice of model organisms in evo-devo

Study of the model organisms of developmental biology was crucial in establishing evo-devo as a new discipline. However, it has been claimed that this limited sample of organisms paints a biased picture of the role of development in evolution. Consequently, judicious choice of new model organisms is necessary to provide a more balanced picture. The challenge is to determine the best criteria for choosing new model organisms, given limited resources.     

水域生态学中生物稳定同位素样品采集、处理与保存

稳定同位素分析技术由于能够刻画复杂的食物网结构并追踪食物网中的能量流而成为水域生态学研究中的重要手段。但是当水生生物样品采集、处理和保存过程中存在不确定性时, 营养关系分析中的同位素结果可能会产生误导性解释。文章采用数据模拟分析和文献总结的方法, 研究了水域生态系统中样品采集、处理和保存对于稳定同位素的影响, 概括性地建议了水域生态系统中适合应用稳定同位素分析技术开展生态学研究的样品采集、处理和保存的注意事项。但今后仍需进一步评估样品采集、处理和保存对稳定同位素比值的影响效果, 确定化学动力学在水生生物样品采集、处理和保存中的作用, 以进一步完善水生生物样品的采集、处理和保存稳定同位素生态学研究规范。     

Improved Microautoradiographic Method to Determine Individual Microorganisms Active in Substrate Uptake in Natural Waters

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with ³H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (³H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.     

Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species

High‐throughput DNA sequencing facilitates the analysis of large portions of the genome in nonmodel organisms, ensuring high accuracy of population genetic parameters. However, empirical studies evaluating the appropriate sample size for these kinds of studies are still scarce. In this study, we use double‐digest restriction‐associated DNA sequencing (ddRADseq) to recover thousands of single nucleotide polymorphisms (SNPs) for two physically isolated populations of Amphirrhox longifolia (Violaceae), a nonmodel plant species for which no reference genome is available. We used resampling techniques to construct simulated populations with a random subset of individuals and SNPs to determine how many individuals and biallelic markers should be sampled for accurate estimates of intra‐ and interpopulation genetic diversity. We identified 3646 and 4900 polymorphic SNPs for the two populations of A. longifolia, respectively. Our simulations show that, overall, a sample size greater than eight individuals has little impact on estimates of genetic diversity within A. longifolia populations, when 1000 SNPs or higher are used. Our results also show that even at a very small sample size (i.e. two individuals), accurate estimates of F_ST can be obtained with a large number of SNPs (≥1500). These results highlight the potential of high‐throughput genomic sequencing approaches to address questions related to evolutionary biology in nonmodel organisms. Furthermore, our findings also provide insights into the optimization of sampling strategies in the era of population genomics.     

Morphological variation of species through time

Ten measurements, taken from each of 700 shells or four biologically distinct shallow marine gastropod species, were used to define the appropriate phenotypes in multidimensional space. Canonical discriminant analysis was performed on the data and a set of allocatory rules was derived. These allocatory rules, derived from extant specimens, were than applied to 644 fossil specimens of three of these biological species. Fossil individuals occupy the appropriate phenotypic space as defined by their modern descendants. The variation of fossil sample means about the modern means is illustrated. This variation is in the form of oscillations around the modern mean values and is correlated with climate. The distinction between taxonomic and biological species is discussed. The results of a number of previous studies are re-examined in the light of this discussion. It is argued that biological groupings can only be reliably determined when the appropriate data are available for extant organisms. Extant organisms, which have good fossil records, should therefore from the basis of paleontological evolutionary studies.     

Rapid electrochemical detection and identification of catalase positive micro-organisms

The rapid detection and identification of bacteria has application in a number of fields, e.g. the food industry, environmental monitoring and biomedicine. While in biomedicine the number of organisms present during infection is multiples of millions in the other fields it is the detection of low numbers of organisms that is important, e.g. an infective dose of Escherichia coli O157:H7 from contaminated food is less than 100 organisms. A rapid and sensitive technique has been developed to detect low numbers of the model organism E. coli O55, combining Lateral Flow Immunoassay (LFI) for capture and amperometry for sensitive detection. Nitrocellulose membranes were used as the solid phase for selective capture of the bacteria using antibodies to E. coli O55. Different concentrations of E. coli O55 in Ringers solution were applied to LFI strips and allowed to flow through the membrane to an absorbent pad. The capture region of the LFI strip was placed in close contact with the electrodes of a Clarke cell poised at +0.7 V for the detection of hydrogen peroxide. Earlier research identified that the consumption of hydrogen peroxide by bacterial catalase provided a sensitive indicator of aerobic and facultative anaerobic microorganisms numbers. Modification and application of this technique to the LFI strips demonstrated that the consumption of 8 mM hydrogen peroxide was correlated with the number of microorganisms presented to the LFI strips in the range of 2 x 10(1)-2 x 10(7) colony forming units (cfu). Capture efficiency was dependent on the number of organisms applied and varied from 71% at 2 x 10(2) cfu to 25% at 2 x 10(7) cfu. The procedure was completed in less than 10 min and could detect less than 10 cfu captured from a 200 microl sample applied to the LFI strip. The approached adopted provides proof of principle for the basis of a new technological approach to the rapid, quantitative and sensitive detection of bacteria that express catalase activity.